The first critical step of HIV-1 infection is fusion of viral and target cell membranes. Viral attachment and membrane fusion are mediated by the viral envelope glycoprotein upon engagement with cellular receptors (Harrison (2008) Nat. Struct. Mol. Biol. 15:690; Wyatt and Sodroski (1998) Science 280:1884). The envelope protein is synthesized as a precursor, gp160, which trimerizes and undergoes cleavage into two, non-covalently-associated fragments: the receptor-binding fragment gp120 and the fusion fragment gp41 (Allan et al. (1985) Science 228:1091; Veronese et al. (1985) Science 229:1402). Three copies of each fragment make up the mature viral spike, which constitutes the sole antigen on the virion surface. Sequential binding of gp120 to the primary receptor CD4 and co-receptor (e.g. CCR5 and CXCR4) induces large conformational changes which then trigger dissociation of gp120 and a cascade of refolding events in gp41 (Harrison, Supra; Harrison (2005) Advances in Virus Research 64:231). Gp41, with its C-terminal transmembrane segment inserted in the viral membrane, is folded into a pre-fusion conformation within the precursor, gp160. Cleavage between gp120 and gp41 makes this pre-fusion conformation metastable with respect to a rearranged, post-fusion conformation. When triggered by the binding of gp120 to the co-receptor, the N-terminal fusion peptide of gp41 translocates and inserts into the target cell membrane. The extended conformation of the protein, with the fusion peptide inserted into cell membrane and the transmembrane anchor in the viral membrane, is referred to as the “pre-hairpin intermediate” (Chan and Kim (1998) Cell 93:681). The pre-hairpin intermediate can be targeted by T-20/Enfuvirtide, the first approved fusion-inhibiting antiviral drug, as well as by certain broadly neutralizing antibodies (Kilby and Eron (2003) New Engl. J. Med. 348:2228; Wild (1992) Proc. Natl. Acad. Sci. USA 89:10537; Frey et al. (2008) Proc. Natl. Acad. Sci. USA 105:3739). Subsequent rearrangements involve folding back of the C-terminal heptad repeat 2 (HR2) region of gp41 into a hairpin conformation, creating a six-helix bundle, which places the fusion peptide and the transmembrane segment at the same end of the molecule (Chan et al. (1997) Cell 89:263; Weissenhorn et al. (1997) Nature 387:426). This irreversible refolding of gp41 effectively brings the two membranes together. During the fusion process, gp41 exhibits at least three distinct conformational states: the pre-fusion conformation, an extended, pre-hairpin intermediate, and the post-fusion conformation.
HIV-1 infected patients typically generate strong antibody responses to the envelope glycoprotein, but most of these antibodies are either non-neutralizing or strain-specific, and many recognize epitopes occluded on mature trimeric spikes or epitopes located in the highly variable loops. Extensive glycosylation, sequence diversity, and receptor-triggered conformational changes and epitope masking pose great challenges to generation of broadly reactive neutralizing antibodies (NAbs) (Richman et al. (2003) Proc. Natl. Acad. Sci. USA 100:4144; Kwong et al. (2002) Nature 420:678; Wei et al. (2003) Nature 422:307). Some patient sera show broadly neutralizing activity, but immunogens that can induce such antibody responses have remained elusive (Stamatatos et al. (2009) Nat. Med. 15:866). Nevertheless, a number of broadly reactive neutralizing monoclonal antibodies (mAb) have been isolated that recognize regions of the HIV-1 envelope glycoprotein. Some are located on gp120: the CD4 binding site (CD4bs), the V2 and V3 loops and the carbohydrates on the outer domain of gp120 (Wu et al. (2010) Science 329:856; Zhou et al. (2010) Science 329:811; Walker et al. (2009) Science 326:285; Trkola et al. (1996) J. Virol. 70:1100; Burton et al. (1994) Science 266:1024; Hioe et al. (2010) PLoS One 5:e10254; Zolla-Pazner and Cardozo (2010) Nat. Rev. Immunol. 10:527). Additional neutralizing antibodies target regions on gp41 adjacent to the viral membrane and are called the membrane proximal external region (MPER; residues 662-683 (HXB2 numbering)) (Stiegler et al. (2001) AIDS Res. Hum. Retroviruses 17:1757; Muster et al. (1993) J. Virol. 67:6642; Zwick et al. (2001) J. Virol. 75:10892).
Gp41 also induces non-neutralizing antibodies which are much more abundant in patients than neutralizing ones. The non-neutralizing antibodies have been classified into two groups based on the location of their epitopes. Cluster I antibodies react with the immunodominant C-C loop of gp41 (residues 590-600), and cluster II antibodies recognize another immunodominant segment (residues 644-663) next to the MPER (Xu et al. (1991) J. Virol. 65:4832). Members in the latter group can bind HIV-1 gp41 with high affinity, but have weak or no neutralizing or antiviral activities (Hioe et al. (1997) Int. Immunol. 9:1281; Holl et al. (2006) J. Virol. 80:6177). The prototype of this group includes mAbs 98-6, 126-6, 167-D, 1281 and 1379, isolated by immortalizing plasma B cells from HIV-1 positive patients (Xu et al., Supra; Gorny et al. (1989) Proc. Natl. Acad. Sci. USA 86:1624; Gorny et al. (2000) Virology 267:220; Pinter et al. (1989) J. Virol. 63:2674). As the conformation of these envelope preparations has not been fully assessed, it remains uncertain which conformation(s) of gp41 the cluster II mAbs recognize and why they are incapable of blocking HIV-1 infection, as do the MPER-directed neutralizing antibodies.